The basis of gene amplification in PALA-resistant hamster cells which overproduce carbamyl-P synthetase, asparate transcarbamylase and dihydro-orotase (as a multifunctional protein) and the corresponding mRNA will be investigated, using cloned cDNA's derived from the purified mRNA and cloned restriction fragments of genomic DNA. The structures of the mRNA and genomic DNA will be compared, and we will do experiments with cells in which we attempt to alter the frequency with which PALAr mutations arise. We will use specific antibodies to the SV40 proteins VP3 and T antigen to investigate further their in vivo functions by determining their locations on SV40 chromatin, and also determining the nature and role of post translational modification of these proteins. We will explore the function of pppA2'p5'A2'p5'A as an effector for a nuclease concerned with regulation of mRNA levels in mammalian cells, and try to elucidate the details of this regulatory mechanism.